We report here differential sputum mediator profiles between groups of severe asthmatic patients stratified by airway wall geometry, with more marked differences between groups when patients were stratified further by sputum neutrophil counts. Some of the differences were closely related to the differential sputum cell counts such as sputum MMP9 and IL-1β, which were elevated in those groups with a high neutrophil count and sputum ICAM-1 was elevated in those subjects with a low sputum neutrophil count independent of airway geometry . However, other differences were observed that were dependent upon both stratification by the sputum neutrophil count and airway geometry namely CCL11, IL-1α and fibrinogen. Sputum CCL11 concentration was elevated in subjects with a low sputum neutrophil count and high luminal and total area, whereas sputum IL1α was increased in subjects with a high sputum neutrophil count and low total area. Sputum fibrinogen was elevated in those subjects with luminal narrowing independent of sputum cellular differential, but was also increased in subjects with neutrophilic inflammation without luminal narrowing. These findings support the view that there are differential sputum mediator profiles related to airway geometry.
Several sputum mediators were associated more with neutrophilic inflammation than the pattern of airway geometry. Two of these mediators IL-1β and MMP-9 are observed in subjects with COPD and are reported to be associated with airway bacterial colonisation and neutrophilic inflammation. Interestingly, Matsumoto et al. observed an inverse relationship between sputum MMP-9 and%WA which was consistent with our observation of a low MMP-9 in the high luminal area, non-neutrophilic group. In contrast, sputum MMP9 was elevated in our study in those with neutrophilic inflammation independent of luminal area . IL-1β and MMP-9 promote subepithelial fibrosis, subepithelial collagen deposition and elastin fibre disruption in asthma and in COPD are associated with many of the histopathological features of emphysema [29–32]. This may suggest a phenotype of remodelling associated more with ‘scaffold destruction’ leading to airway dilatation from loss of airway wall integrity as opposed to a predominant airway thickening. Indeed, the group with high total and luminal area were associated with increased qualitative evidence of bronchiectasis and frequent exacerbations. Although this process has relevance to those subjects with luminal dilatation, these mediators were also increased in subjects with neutrophilic inflammation and luminal narrowing. This suggests either that in these subjects an alternative inflammatory process is driving the airway narrowing or that there were changes that occurred earlier in the lung development of these subjects that led to either airway narrowing in early development or a predisposition to airway narrowing in the natural history of the disease.
Fibrinogen was also elevated predominately in those with neutrophilic inflammation, but was increased in subjects without neutrophilic inflammation with luminal narrowing and low RB1 total area. Fibrinogen is an acute phase soluble plasma glycoprotein, synthesised primarily in the liver and converted by thrombin into fibrin during blood coagulation. It is emerging as a promising systemic biomarker in COPD that shows relationships with disease severity, progression and mortality . There are studies that link its presence to microvascular leakage from plasma exudation, which occurs during remodelling or neurogenic inflammation in asthma [34, 35] and may contribute to pathophysiological features of airway hyperresponsiveness . Fibrinogen leakage exudes into the extravascular compartment and interacts with neutrophils causing their sequential activation and promoting their survival by reducing apoptosis . Sputum fibrinogen is therefore likely to be increased in subjects with airway wall oedema, which may contribute to the luminal narrowing in subjects independent of sputum cell differential. Interestingly, it was not increased in subjects without a sputum neutrophilia and without high RB1 suggesting that in this group airway wall oedema and increased vascularity may not play a prominent role. This needs to be confirmed by examination of the bronchial wall in this group of subjects. Intriguingly, this group also had elevated sputum CCL11 and the highest sputum eosinophil count, albeit non-significant, suggesting that although eosinophilic inflammation may be important in driving remodelling and luminal narrowing in some subjects it is also associated with normal airway geometry. Whether this represents a group of patients protected from airway remodelling or at an earlier stage of the natural history needs to be further explored.
In contrast to sputum IL-1β, sputum IL1α was not closely related to neutrophilic inflammation but was increased in those subjects with low RB1 luminal area increased and in those subjects with both low RB1 total area and neutrophilic inflammation. Recent evidence has implicated IL-1α as an important link between the innate immune system and allergic sensitisation TLR4 activation induces IL-1α release from epithelial cells, which acts in an autocrine manner to release GM-CSF and IL-33 . IL-1α is a potent activator of fibroblasts  and might play a role in fibroblast activation and hyperplasia in asthma. In atherosclerosis  IL-1α has also been implicated in the plaque stability and outward vessel remodelling. Whether IL-1α plays a similarly important role in differential changes in airway geometry warrants further study.
One major criticism of our study is the cross-sectional design, which limits our interpretation of the dynamic relationship between the mediator profiles and airway remodelling. For example, it is uncertain whether the changes observed by CT imaging reflect progressive remodelling over many years as a consequence of airway inflammation or represent changes in airway geometry that were established in early lung development . For example, there were small differences in the age of onset of disease and current age, albeit not disease duration, in subjects in the CT defined subgroups. This effect of time is particularly important when considering differential mediator profiles between subjects in whom the airway wall has remodelled towards the airway lumen versus those that have either maintained normal luminal patency or have remodelled away from the lumen and have airway dilatation. To address this question either longitudinal studies of the natural history of remodelling are required or studies of interventions that might impact in the short term upon remodelling such as bronchial thermoplasty [42, 43]. Importantly, luminal area in the severe asthma group is decreased compared to controls such that even the high luminal area group described here do not represent bronchial dilatation compared to health. A further criticism is that sputum analysis reflects a composite measure of the proximal airways rather than a specific lobe whereas the CT imaging here focussed upon the dimensions of the RB1. Although a more comprehensive volumetric analyses the entire proximal airway tree would be informative we and others have reported that the RB1 geometry has good correlations with other proximal airways . Therefore, we are confident that this is not a major limitation of this study. The sputum samples were processed with the mucolytic DTT, which affects the recovery of a number of mediators in particular Th2 cytokines. The scope of the mediators measured here are therefore hampered by this shortcoming, but none the less we have measured a comprehensive panel of biomarkers and are confident this reflects many aspects of the inflammatory profile in the airway of these subjects. Sputum analysis is restricted to sampling to the airway lumen and does not necessarily reflect airway inflammation in the airway wall. Large studies of the relationship between airway wall remodelling determined by bronchial biopsies and CT imaging are underway and the findings are eagerly awaited.
In conclusion, severe asthma is a complex heterogeneous disease and the interactions between airway inflammation and remodelling are poorly understood. Here we have demonstrated that sputum mediator profiling reveals a number of associations between airway geometry and sputum differential cell counts and mediator concentrations. Whether these findings reflect specific and consistent underlying biological phenotypes with predictable natural histories and the potential for stratified medicine approaches needs to be further investigated.